Importance of lactate dehydrogenase (LDH) and monocarboxylate transporters (MCTs) in cancer cells

Abstract Background In most regions, cancer ranks the second most frequent cause of death following cardiovascular disorders. Aim In this article, we review the various aspects of glycolysis with a focus on types of MCTs and the importance of lactate in cancer cells. Results and Discussion Metabolic changes are one of the first and most important alterations in cancer cells. Cancer cells use different pathways to survive, energy generation, growth, and proliferation compared to normal cells. The increase in glycolysis, which produces substances such as lactate and pyruvate, has an important role in metastases and invasion of cancer cells. Two important cellular proteins that play a role in the production and transport of lactate include lactate dehydrogenase and monocarboxylate transporters (MCTs). These molecules by their various isoforms and different tissue distribution help to escape the immune system and expansion of cancer cells under different conditions.

glycolysis by-product that mainly produced in the absence of oxygen in the body is one of the adaptive proceedings of tumor cells for survival and further progression. 8

| METHODOLOGY
In this study, data on various aspects of glycolysis with a focus on types of MCTs and lactate in cancer cells were found using PubMed, Scopus, and the Google Scholar database. The Internet searches were done to find published manuscripts with the keywords Cancer metabolism, Monocarboxylate transporters, lactate. All English language articles were found and read independently by two individuals. Literature search strategy for data in tables including inclusion/exclusion criteria and results was provided in Supporting Information: Figure S1.

| Advantages of aerobic glycolysis pathway for tumor growth
Even in the presence of ample oxygen, cancer cells demonstrate a distinctive form of cellular metabolism characterized by high levels of glucose uptake and increased conversion of glucose to lactose via the glycolytic pathway (fermentation). This phenomenon called the Warburg effect and known anaerobic glycolysis, has been recognized for many years. Warburg metabolism is not cancer-specific, but instead is a general property of growing cells that is exploited by cancer cells. Aerobic glycolysis provides rapidly dividing tumor cells with metabolic intermediates that are needed for the synthesis of cellular components, whereas mitochondrial OXPHOS does not.
The aerobic glycolysis pathway provides several advantages for tumor cells growth as below. Using aerobic glycolysis, the cancerous cell can survive in variable levels of oxygen pressure (due to unstable hemodynamics of blood vessels) and produce ATP (this condition is associated with risk of death for OXPHOS-dependent cells). 13 Tumor cells produce bicarbonate and lactate. These acids make the microenvironment suitable for invasive tumor cells and suppress the immune system. 14 and MCT2) and used to pyruvate produce. 15 The most important advantage is using the glycolysis-pathway mediators for anabolic reactions (e.g., use of glucose 6-phosphate for the synthesis of glycogen and ribose 5-phosphate and nicotinamide adenine dinucleotide phosphate [NADPH], dihydroxyacetone phosphate for the synthesis of triglycerides and phospholipid, pyruvate for the synthesis of alanine and malate). 17 The exploitation of the pentose phosphate pathway (PPP) as one of the glycolysis branching commonly increases in tumorgenesis. Moreover, some of the major enzymes of this pathway, such as transketolase 1 and transaldolase, have shown increased expression in many cancers. [18][19][20][21] Also, one of the reasons for reducing the use of the Krebs cycle is some of the products of this cycle, such as nicotinamide adenine dinucleotide hydrogen (NADH) and adenosine triphosphate (ATP), which are the main inhibitors of glucose metabolism. 22

| Absorption of glucose and amino acids (increased demand for nitrogen)
Glucose and glutamine (unnecessary amino acid with two reduced nitrogen atoms) are the most important and essential precursors for the biosynthesis of cellular materials). Due to increased carbon consumption in biosynthetic pathways and growth signals, the cell's need for nitrogen increases. Transcription factors such as C-MYC and E2F increase glutamine uptake. 22 Lack of glutamine leads to cell cycle arrest in the S-phase in some cells. 23,24 Embryonic stem cells, luminal cells of breast cancer, 25,26 and human glioblastoma tumors continue their growth and proliferation, even in the absence of glutamine in their microenvironment, which represents the glutamine production by these cells de novo. 27 It also has been seen an increase in the expression of glutamine synthase (GS) enzyme in some cancers. 28

| Opportunity to absorb nutrients
The Ras or c-Src mutation enables cells to uptake released amino acids by the lysosomal degradation of extracellular proteins. 29 Macropinocytosis is also stimulated by Ras-and c-Src-driven actin cytoskeleton remodeling. 22 Moreover, soluble extracellular proteins and free amino acids can be absorbed into cancer cells by entosis or phagocytosis. 30,31 Cancer cells, in addition to the absorption of fatty acids from plasma, can induce the release of stored lipids in adjacent normal cells. 22 Furthermore, increased expression of monoacylglycerol lipase (MAGL) and lipoprotein lipase in some of the cancer cells has been related with the invasion of them. 32,33 At the surface of the metastatic ovarian cancer cells, the expression changes of fatty acid binding protein 4 (FABP4) enable these cells to absorb fatty acids from the omental fat adipocytes. 34 However, fatty acid de novo biosynthesis in normal cells is very low (except lipogenic tissues such as the liver, adipose tissue, and mammary epithelium during lactation). 35,36

| Glycolysis pathway and cancer cells
The result of the anaerobic metabolism of glucose in cancer cells that reside far from vessels is lactate production, which releases from these cells and entered into the intercellular spaces. This metabolite is entered to near vasculature cancer cells from intercellular space and provides their energy supply. 9 The glycolysis pathway in cancer cells allows them to consume most of the cell glucose storage and convert it to lactate. Lactate stimulates and enhances cancer cell growth and proliferation. Therefore, it is imperative to recognize and survey the genes involved in this pathway, especially the genes that their product plays an important role in lactate metabolism. 54 For example, some cancer cells such as cells in glioblastoma use aerobic glycolysis pathway for energy production even in the presence of oxygen (Warburg effect) ( Figure 1). 55 As noted earlier, a large quantity of lactate and proton is produced due to the increasing glycolysis. This extra proton led to an acidic space inside the cell, which can induce normal cell apoptosis under normal conditions. 56 However, this extra proton is sent out or used by cancer cells through different mechanisms 57 such as using the family of MCTs, 58 H/Na exchanger (NHE), 59 V-ATPase pump, 60,61 carbonate anhydrase enzyme, 16 and the conversion of glutamate to gamma aminobutyrate. 62 Since this acidity increases the cancer cells invasion potentials as well as drug resistance; therefore, these molecules are overexpressed in cancerous cells than normal cells. 55 Moreover, not only lactate acts as a signal to stimulate angiogenesis but also is an immunosuppressive agent. 63 There are two main checkpoints for regulating the production and transport of lactate in the cell included lactate dehydrogenase (LDH) and the family of MCTs. LDH has an important role in the conversion of pyruvate to lactate, and MCTs are involved in the transport of lactate into and out of the cells. 8 These monocarboxcylates, like lactate, can be transferred to other cancer cells, or use for OXPHOS.

| LDH
LDH is produced in normal cells such as the brain, heart, liver, kidney, skeletal muscle, red blood cells, and lungs. 64,65 However, LDH is overexpressed in some conditions such as hypothyroidism, anemia, meningitis, myocardial infarction, acute pancreatitis, acquired immunodeficiency syndrome (AIDS), liver/pulmonary disease, as well as in most of cancerous cells. Therefore, it can be used as a cancer cell marker or as a therapeutic option for cancer. 66 This enzyme is found in different organisms. LDH converts pyruvate to lactate when oxygen is absent or trace and it performs the reverse reaction in the Cori cycle in the liver. 64 Therefore, LDH is an enzyme with two forward and reverse functions. Structurally, this enzyme is a tetrameric enzyme composed of two major subunits (A, B), which are encoded by two the Lactate dehydrogenase A (LDHA) (11p15) and LDHB (12p12) genes. These two subunits produce five isoenzymes. 65 • Activating the Akt signaling pathway • Decrease in expression of CPT1A (carnitine palmitoyltransferase) enzyme • Changing in this pathway is one of the most common events in human cancers • Increasing of glucose uptake, lipid synthesis, and glycolysis [159] Akt ( Figure 1) • Increase in regulation of fatty acid production • Increasing of expression of FAS enzyme • Activating of mTOR pathway • Increase cell growth and invasion [160] ErbB2 (Her2) • Increasing the synthesis of fatty acid and cell growth (by increasing the regulation of acetyl-CoA carboxylase alpha and fatty acid synthase) • mTOR, Akt, and PI3K signaling pathways activating [161] PD-L1 • Its expression regulated by oncogenic transcription factors, such as C-MYC and HIF • In cancer cells of the ovary and melanoma, the high expression of PD-L1 promotes cell growth and autophagy through the mTOR pathway. • The PD-L1 connects directly to ITGB4 and activates the AKT/GSK3β and the ITGB4/1/ SNAI3 SIRT signaling pathways • The high expression of PD-L1 and ITGB4 in cervical cancer was significantly associated with metastasis and poor prognosis of lymph nodes [162][163][164][165][166][167][168][169][170][171][172] CAV1/caveolin 1 • Increasing of aerobic glycolysis (by SLC2A3/GLUT3 activating) [173,174] T A B L E 1 (Continued) • Inducing of glycolysis and role in development, proliferation, invasion, and metastasis of tumor cells [175,176] Ecdysoneless • Glycolysis regulation and role in metastasis and proliferation of tumor cells [177] FAK • Glucose uptake stimulation and shifting its consumption from oxidative phosphorylation to glycolysis • Glycolysis and tumorgenesis induction [178] GRP78 • Stimulated by HIF-1a • Stimulation of glucose Consumption, angiogenesis, and metastasis • Induction of autophagy, resistance to apoptosis, and escape from the immune system • Reduction of glycolysis and shifting glucose consumption from glycolysis to Krebs cycle • Decrease the expression of PKM2 and increase the expression of mitochondrial PDHA and B PDHB and Glut-1 [179] KSHV • Induction of aerobic glycolysis and angiogenesis by HIF-1a and PKM2 • Increase in VEGF levels [180] LMP1 • Stimulation of aerobic glycolysis, cell growth, metastasis, invasion, and angiogenesis • Increasing of glucose and glutamine absorption and lactate production • Increase in expression of genes such as PDHK1, FGFR1, c-Myc, HIF-1α [181] P2X7R • Inhibition of pyruvate dehydrogenase • Akt/PKB and HIF-1α pathways inducing • Increasing of glycogen intracellular storage • Increase regulation of Glut1, G3PDH, PFK, PKM2, and PDHK1 [182] Skp2 • Akt activation • Role in metastasis, poor prognosis, and disease progression • Increase in glucose uptake and consumption (by Glut1 expression increasing) [183] BIRC5/Survivin • Increase aerobic glycolysis and drug resistance • Apoptosis inhibition (BCL2L11/Bim inhibition) • Reduced mitochondrial respiration [184] Wnt/β-catenin • Increase in aerobic glycolysis, angiogenesis, and cell growth [152] p53 ( Figure 1) • Role in glucose and fatty acids metabolism (inhibition of glycolysis and glucose transporters, negative regulation of phosphoglycerate mutase and AKT) • Stimulation of the catabolic pathways (i.e., fatty acids oxidation) • Increase ROS production [185][186][187][188] Fumarate hydratase (fumarase) • Helping to HIF-1a degradation • (Fumarate accumulation inhibits HIF1a prolyl hydroxylase) [189] abhd5 (other name: CGI-58) • Cell growth reduction • inhibition aerobic glycolysis and EMT • The AMPKα-p53 pathway inducing [190] GRIM-19 • Aerobic glycolysis reducing • Decreasing of cell growth and proliferation • Increase P53 activity • Decrease Stat3 and HIF-1a activity • Increase oxygen consumption [191] HSP40 • Binds to pyruvate kinase M2 and destroy it that resulted to reducing glucose consumption and lactate production • Reduce growth and cell proliferation [192] KLF4 • Reducing of aerobic glycolysis and decreasing of cell growth and proliferation • LDHA expression reduction [193] (Continues) HATAMI ET AL.  (Figure 1).

| MCTs
Monocarboxylate is referred to the compounds such as lactate, pyruvate, ketone bodies, and thyroid hormones that play an important role in the energy metabolism of different tissues (i.e., skeletal muscle, heart, brain, and blood cells). 69 In patients with (rs1049434) polymorphism, a reduction of 35%-40% in lactate transmission in erythrocytes has been observed. 84 Two polymorphisms (rs10506398 and rs10506399) in MCT2 have been associated with reduced sperm count and infertility in men. 76,85 Polymorphism (rs2242206) in MCT-9 causes lysine to be transformed into threonine at position 258, which is associated with renal overload gout. 86 A nonsense mutation in the MCT-12 gene, which causes Q215X amino acid mutation, has been observed in patients with cataract and increased levels of urinary glucose. 87 In
Hyaluronan is another protein that interacts with MCTs. 109 Hyaluronan is an extracellular polysaccharide that is present in most tissues and has an instructive, cell signaling function in addition to its structural role. [110][111][112][113] The concentration of hyaluronan is more in malignant cells than in normal tissues. 109,114,115 Hyaluronan binds to various cell surface receptors such as CD44, Rhamm, TLR1/4, Hare and LYVE-1. [110][111][112][113] Among these receptors, CD44 is the most The metabolic changes in the malignant cells. After conversion of glucose to pyruvate and entrance into the oxidative phosphorylation pathway, pyruvate converts to acetyl CoA. Acetyl CoA by entering the mitochondria (Krebs cycle) generates energy. In most of the malignant cells, after pyruvate generation, these cells use the aerobic glycolysis pathway (Warburg effect (pyruvate by lactate dehydrogenase, LDH, enzymes convert into lactate and proton)). Produced lactate has been sent out of the cell by monocarboxylate transporters (MCTs) and has been shown that play important role in increases the angiogenesis, migration, and metastasis of cells, escape from the immune system, extracellular acidosis, reduction of the monocytes migration, secretion of cytokines and activation of T lymphocytes. Moreover, with the help of these monocarboxylates transporter, lactate can reenter to the cell and again converts to pyruvate by the LDH and generates energy. With the increase in glycolysis, proton production also increases, and these excess protons through proton pumps such as MCT, H/Na exchanger, ATP synthase, pepT, V-ATPase are transmitted into extracellular space and causes acidosis in the microenvironment of the cancer cells. Activation of oncogenes, such as MYC, HIF, RAS, and Akt, and interaction of them with different molecules in the cell, is also the major cause of the biogenic changes in tumor cells.  [72,75,119,[196][197][198][199][200][201][202][203] MCT-2 SLC16A7 12q14
known. The binding of this receptor to hyaluronan regulates signaling pathways such as apoptosis and drug resistance to anticancer agents. 109 Emmprin as an essential ancillary protein for MCTs by stimulating the production of hyaluronan plays an important role in the invasion and division of cancer cells. 112,116,117 Studies have shown that lactate produced in the glycolysis pathway stimulates the production of hyaluronan and the expression of CD 44 in fibroblasts and melanoma cells. 53,118 The interaction of Emmprin-CD44 is one of the main causes of malignancy and drug resistance in cancer cells. 109 Several studies have been conducted on the MCTs that the history of major studies and their results from 1999 to 2018 is shown in Table 4.

| Diagnostic and therapeutic applications of metabolic changes in the cancer cells
Cancer is a disease that is known to alter cellular metabolism; therefore, metabolomics can play a major role in the early detection and diagnosis of cancer and in the evaluation of medical interventions and therapies to cancer. 132 It has been established that aerobic glycolysis increases in cancer and this is known as the "Warburg effect." 133 The ultimate goal of most metabolomics cancer studies is to discover cancer-specific diagnostic, prognostic, or predictive biomarkers for a patient. Metabolomics research is being used to discover diagnostic cancer biomarkers in the clinic, to better understand its complex heterogeneous nature, to discover pathways involved in cancer that could be used for new targets and to monitor metabolic biomarkers during therapeutic intervention. 134  (2) Transport undependent Na and proton • This protein is involved in carnitine (as an antioxidant, with increasing the activity of nitric oxide enzyme, decreasing the activity of the arginase enzyme and the level of ornithine, as well as reducing the side effects of tamoxifen, has an important role in improving the prognosis) transmission [125,126] 2014 MCT4 • Expression of MCT4 is increased in triple-negative breast cancer and associates independently with clinical consequence [127] 2016 MCT1 • In glioblastoma cell lines, its effect on cell proliferation and cell migration was investigated • Increased expression of MCT1 in cancer stem cells was shown • Inhibition of this gene was also proposed as a therapeutic target [55] 2017 MCT1 and 4 • Increased expression of MCT1 and 4 in different breast cancer cell lines, blood and tissues of patients with breast cancer has been seen • The study suggested that MCT1 and 4 as can be considered as therapeutic targets [128] 2017 MCT8 • In thyroid cancer, MCT8 expression is reduced • Could be considered as a potential thyroid differentiation marker • In previous studies, MCT8 expression in the brain, heart, kidney, liver, placenta, and testis was examined [129] 142 Currently, AZD3965 is being evaluated in a phase I dose-ranging study. 139

| CONCLUSION
Changed energy metabolism is emerged as one of the very important cancer biochemical fingerprint which could be introduced as a cancer hallmark. These metabolic events are characterized by preferential dependence on glycolysis for the production of energy in an oxygenindependent condition. Glycolysis is one of the major underlying energy-related processes, which play critical roles in the initiation, and progression of all malignancies. Pyruvate and lactate are the main products of glycolysis. Given that, some cellular proteins (i.e., LDH and MCTs) involved in the production and transport of lactate.
Considering the vital role of LDH and MCTs in the metabolism of cancer cells, these molecules can be investigated as a target for treatment or as markers in cancer diagnosis in future studies.
Mounting evidence confirmed that these molecules could contribute to escape cancer cells from the immune system and promote the expansion of tumor cells under distinct conditions.